OP0095 SINGLE-CELL RNA SEQUENCING REVEALS POTENT ANTI-INFLAMMATORY AND ANTIFIBROTIC ACTIVITIES OF DIMETHYL-ALPHA-KETOGLUTARATE ON EXPLANTED SKIN FROM PATIENTS WITH SYSTEMIC SCLEROSIS
Authors: B. Burja, D. Paul, R. Gerber, S. G. Edalat, M. Elhai, E. Pachera, R. S. Zingg, F. M. Pramotton, S. F. Madsen, K. Buerki, G. Costanza, M. Whitfield, A. C. Bay-Jensen, S. Sodin-Šemrl, M. Tomsic, G. Kania, H. Rehrauer, O. Distler, Z. Rotar, M. Robinson, K. Lakota, M. Frank Bertoncelj
Journal: Annals of the Rheumatic Diseases (2022)

Background Activated fibroblasts are the main drivers of skin fibrosis in SSc. We have recently identified dimethyl alpha-ketoglutarate (dm-aKG) as a potential repressor of myofibroblast differentiation and profibrotic activity in cultured skin fibroblasts. Objectives To further analyse the clinical translation of our findings by investigating the antifibrotic capacity of dm-aKG on explanted skin biopsies from SSc patients. Methods We cultured forearm punch skin biopsies from SSc patients (n=10) for 24h ex vivo in the presence/absence of 6 mM dm-aKG. Thereafter, skin biopsies (n=4) were dissociated into single cells using a combined mechanical-enzymatic dissociation protocol, followed by single cell (sc)RNA-seq library preparation (10x Genomics) and sequencing (Illumina, NovaSeq6000, 50,000 reads/cell). We mapped the scRNA-seq reads to the reference genome GRCh38.p13 and analysed the data with R/Bioconductor tools. We deconvoluted cell types in bulk skin transcriptomes from SSc cohorts (GSE: 45485, 59785, 9285, 32413) using human skin scRNA-seq data 1 . The secretion of IL-6, procollagen-1, PRO-C1 (N-terminal type I collagen pro-peptide), C1M (MMP-degradation fragment of type I collagen), and fibronectin (FBN-C) from cultured skin (n=10) was measured in supernatants by ELISA. We analysed gene and protein expression in TGFβ-activated healthy and SSc dermal fibroblasts (DF, n=10) treated or not with dm-aKG using qPCR, Western blot and ELISA. Contractile properties of DF were assessed by gel contraction assay. Traction forces generated by DF were determined by reference-free traction force microscopy. Results Dissociated cultured SSc skin exhibited comparable cell yield and viability in the presence (20,203; 89%) and absence (25,280; 93%) of dm-aKG, respectively. scRNA-seq skin analysis included 20,869 high quality single cell profiles segregating into 10 distinct skin cell populations (Figure 1A). This analysis demonstrated decreased proportion of fibroblasts and increased proportion of keratinocytes in dm-aKG treated skin (p&lt;0.05; Figure 1B). Among skin cell types, skin fibroblasts exhibited the largest amount of differentially expressed genes upon dm-aKG treatment (44%, n=779, x-fold&gt;0.5, FDR&lt;0.05), suggesting that these cells are key targets of dm-aKG therapy in SSc skin. We identified inflammatory/cytokine signalling (hub genes IL6, STAT1 ) and extracellular matrix (ECM) organization (hub genes MMP1, ITGB3 ) as top downregulated biological processes in fibroblasts in dm-aKG treated SSc skin (Figure 1C), coinciding with a decreased abundance of proinflammatory skin fibroblast subpopulation. Specifically, these cells were identified as the main source of IL6 (Figure 1D) and were enriched in SSc skin as revealed by deconvolution analysis of skin transcriptomes. Furthermore, dm-aKG reduced the secretion of IL-6, procollagen-1 and C1M, but not pro-C1 and FBN-C, from cultured skin explants. In cultured DF, dm-aKG blocked the inflammatory (IL-6, pSTAT3), profibrotic (aSMA, Fibronectin, Procollagen-1, Pro-C1) and contractile activities, and significantly diminished traction forces exerted by DF on the matrix substrate. Figure 1. scRNA-seq – comparison of untreated and dm-aKG treated paired skin biopsies. (A) UMAP plot with annotated skin cells, (B) differential abundance of main skin cell types, (C) volcano plot of DE genes with top downregulated gene ontology (GO) pathways in dm-aKG treated skin fibroblasts, (D) IL6 expression in untreated (blue) and treated (pink) skin fibroblasts. Conclusion Dm-aKG broadly interferes with inflammatory and ECM organizational activities of skin fibroblasts in culture and in explanted skin from SSc patients. These results confirm that dm-aKG might represent a potential new therapeutic approach for efficient targeting of skin inflammation and fibrosis in SSc. References [1] He H et al. J Allergy Clin Immunol 2020 Acknowledgements This work was supported by a research grant from FOREUM Foundation for Research in Rheumatology and University Medical Centre Ljubljana. Disclosure of Interests Blaž Burja: None declared, Dominique Paul: None declared, Reto Gerber: None declared, Sam G. Edalat: None declared, Muriel Elhai Speakers bureau: BMS, Elena Pachera: None declared, Rahel S. Zingg: None declared, Francesca Michela Pramotton: None declared, Sofie Falkenløve Madsen: None declared, Kristina Buerki: None declared, Giampietro Costanza: None declared, Michael Whitfield: None declared, Anne-Christine Bay-Jensen: None declared, Snežna Sodin-Šemrl: None declared, Matija Tomsic: None declared, Gabriela Kania: None declared, Hubert Rehrauer: None declared, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, Janssen, Medscape, Consultant of: Abbvie, Acceleron, Alcimed, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, 4P Science, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Prometheus, Roivant, Sanofi and Topadur, Grant/research support from: Kymera, Mitsubishi Tanabe, Boehringer Ingelheim, Ziga Rotar: None declared, Mark Robinson: None declared, Katja Lakota: None declared, Mojca Frank Bertoncelj: None declared.